RT-PCR Detection of Avian Coronaviruses of Galliform Birds (Chicken, Turkey, Pheasant) and in a Parrot

Of the many primer combinations that we have investigated for the detection of avian coronaviruses, two have worked better than any of the others: they worked with the largest number of strains/samples of a given coronavirus and the most species of avian coronavirus, and they also produced the most sensitive detection tests. The primer combinations were: oligonucleotide pair 2Bp/4Bm, which is in a region of gene 1 that is moderately conserved among all species of coronavirus (1); and UTR11-/UTR41+, which are in a highly conserved part of the 3′ untranslated region of avian coronaviruses related to infectious bronchitis virus (2). The gene 1 primer pair enabled the detection of a new coronavirus in a green-cheeked Amazon parrot (Amazon viridigenalis Cassin). In this chapter we describe the use of these oligonucleotides in a one-step (single-tube) RT-PCR, and describe the procedure that we used to extract RNA from turkey feces.